The long-term objective of the proposed research is to determine how antibodies contribute to host defense against intracellular bacteria. Our model system uses the obligate intracellular bacterium Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME). Preliminary studies have confirmed the feasibility of the mouse model to study immunity to E. chaffeensis, and its relevance to human disease. These studies have also revealed that significant immunity could be achieved in susceptible immunocompromised SCID mice after passive transfer of antibodies. Bacterial clearance was observed in some tissues even when antibodies were administered well after infection was established, and repeated antibody administration rescued mice from an otherwise fatal infection. Our preliminary studies have determined that Fc receptors for IgG are critical for antibody effectiveness, and that antibodies do not block bacterial adhesion or entry into cells. These later observations suggest that antibodies actively elicit critical effector functions in host phagocytes. The proposed studies will determine how antibody protects against intracellular infection, and will resolve the effector mechanism(s) involved. The Specific Aims are to 1) Determine where antibodies encounter bacteria and mediate their effects in vivo, 2) Evaluate the role of particular FcgRs and antibody class during the humoral immune response, 3) Determine how inflammatory processes induced upon antibody administration contribute to host defense, and 4) Determine how antibodies, via FcgRs, affect the localization and survival of bacteria inside macrophages. Aim#1 will resolve if the general mechanism of antibody mediated bacterial clearance by determining if antibodies mediate their effects only when bacteria are exposed during intercellular transfer. Aim #2 will utilize several isotype switch variants of protective antibodies and gene targeted mice deficient for Fc receptors to identify which antibodies and receptors are critical for antibody efficacy. Aim #3 will test the hypothesis that cytokines, complement, and other inflammatory mediators elicited upon antibody administration play important roles in host defense. Aim #4 will utilize an in vitro infection model to determine if antibodies promote the fusion of phagosomes with lysosomes, and if antibodies can access bacteria in early endosomal compartments. Antibodies have previously been considered to be of little or no effectiveness during host defense against intracellular bacteria, so the study is significant not only for its relevance to our understanding of immunity to the ehrlichiae, but also because it will contribute to a re-evaluation of the tenet that antibodies are unimportant during intracellular bacterial infection, and a re-consideration of the value of antibodies for the treatment and prevention of diseases caused by these pathogens.